Dimerization of a phage-display selected peptide for imaging of α_vβ₆- integrin: Two approaches to the multivalent effect

The integrin α_vβ₆ is an emerging biomarker for non-small cell lung cancer (NSCLC). An α_vβ₆ -binding peptide was previously selected from a phage-displayed peptide library. Here, we utilize a multivalent design to develop a peptidic probe for positron emission tomography (PET) imaging of α_vβ₆⁺ NSCLC tumors. Multimeric presentation of this peptide, RGDLATLRQL, on a bifunctional copper chelator was achieved using two approaches: Dimerization of the peptide followed by conjugation to the chelator (H₂-D10) and direct presentation of two copies of the peptide on the chelator scaffold (H₂-(M10)₂). Binding affinities of the divalent peptide conjugates are four-fold higher than their monovalent counterpart (H₂-M10), suggestive of multivalent binding. PET imaging using the bivalent ⁶⁴Cu-labeled conjugates showed rapid and persistent accumulation in α_vβ₆⁺ tumors. By contrast, no significant accumulation was observed in α_vβ₆ ^- tumors. Irrespective of the dimerization approach, all divalent probes showed three-fold higher tumor uptake than the monovalent probe, indicating the role of valency in signal enhancement. However, the divalent probes have elevated uptake in non-target organs, especially the kidneys. To abrogate nonspecific uptake, the peptide's N-terminus was acetylated. The resultant bivalent probe, ⁶⁴Cu- AcD10, showed drastic decrease of kidney accumulation while maintaining tumor uptake. In conclusion, we developed an α_vβ₆-integrin specific probe with optimized biodistribution for noninvasive PET imaging of NSCLC. Further, we have demonstrated that use of multivalent scaffolds is a plausible method to improve library selected peptides, which would be suboptimal or useless otherwise, for imaging probe development.